U.S. patent number 4,915,933 [Application Number 06/878,651] was granted by the patent office on 1990-04-10 for mixed complexes as shift and contrast reagents in nmr imaging and spectroscopy.
This patent grant is currently assigned to The University of New Mexico. Invention is credited to Nicholas A. Matwiyoff.
United States Patent |
4,915,933 |
Matwiyoff |
April 10, 1990 |
Mixed complexes as shift and contrast reagents in NMR imaging and
spectroscopy
Abstract
Mixed anionic complexes of the type (MW.sub.m Y.sub.n
Z.sub.p).sup.r- wherein M is a paramagnetic ion; W, Y, and Z are
each different ligands which chelate M; and m+n+p.gtoreq.2 but
preferably less than 5, with the proviso that at least two
different ligands W, Y, Z are present in the complex; are provided
as versatile NMR contrast and shift reagents, especially for
clinical diagnostic imaging and spectroscopic procedures. In an
exemplary embodiment, at least one of the ligands W, Y, Z is
metabolizable by the target tissue, and at least one of the ligands
W, Y, Z is substantially inert; the complex is thus tailorable to
improve both physiological tolerance and tissue specificity of NMR
contrast and shift reagents, while maintaining excellent contrast
and shift effects for reliable and accurate results.
Inventors: |
Matwiyoff; Nicholas A. (Sante
Fe, NM) |
Assignee: |
The University of New Mexico
(Albuquerque, NM)
|
Family
ID: |
25372509 |
Appl.
No.: |
06/878,651 |
Filed: |
June 26, 1986 |
Current U.S.
Class: |
424/9.34;
424/9.36; 424/9.361; 424/9.362; 424/9.363; 424/9.364; 436/173;
436/806; 600/420 |
Current CPC
Class: |
A61K
49/06 (20130101); Y10S 436/806 (20130101); Y10T
436/24 (20150115) |
Current International
Class: |
A61K
49/06 (20060101); A61K 049/00 (); A61B
006/00 () |
Field of
Search: |
;424/9 ;436/173,806
;128/653,654 ;556/83 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Magnetic Resonance Imaging, vol. 2, pp. 107-112, 1984, "Relaxation
Enhancement Using Liposomes Carrying Paramagnetic Species", Caride
et al..
|
Primary Examiner: Warden; Robert J.
Assistant Examiner: Wieder; Stephen C.
Attorney, Agent or Firm: Buttmi; Jean A. Fallow; Charles
W.
Claims
What is claimed is:
1. In a nuclear magnetic resonance diagnostic method of the type
wherein a paramagnetic species is employed in an amount sufficient
to observably alter the magnetic properties of a reference ion in
vivo or in vitro, the improvement comprising employing as the
paramagnetic species an anionic mixed complex of a paramagnetic ion
of the formula:
wherein
M is a paramagnetic ion;
W, Y, and Z are each a different bidentate or polydentate ligand
which is a chelating agent for the ion M;
m+n+p.gtoreq.2 but less than 5, and no more than one of m, n, or p
is zero; and
the complex has an overall negative charge r-.
2. The method of claim 1, wherein the reference ion is a sodium
ion.
3. The method of claim 2, wherein the paramagnetic ion M is
selected from the group consisting of paramagnetic lanthanide ions,
Mn(II), Mn(III), Fe(II), Cu(II) and Cr(III).
4. The method of claim 3, wherein the paramagnetic ion M is Dy(III)
or Gd(III).
5. The method of claim 2, wherein each of the ligands is
independently selected from the group consisting of
aminecarboxylates, Schiff bases, aminecarboxylatephosphonates,
porphryins, cryptates, hydroxamates, polyacetates,
tetraazacyclododecanes, phosphates, phosphonates,
aminephosphonates, C.sub.3 -C.sub.20 -peptides, amino acids, and
salicylic acid, acetoacetic acid, oxalic acid, citric acid,
aspartic acid and esters thereof.
6. The method of claim 2, wherein at least one of the ligands is
metabolizable in vivo.
7. The method of claim 6, wherein the metabolizable ligand is
pyrophosphate; tripolyphosphate; an amino acid; pyridoxal;
desferrioxamine; polyglutamic acid; or acetoacetic acid, oxalic
acid, citric acid, salicylic acid, or an ester thereof.
8. The method of claim 6, wherein the ligand metabolizable in vivo
is preferentially metabolized by cells in a target tissue.
9. The method of claim 6, wherein at least one of the ligands is
metabolically inert in vivo.
10. The method of claim 9, wherein the inert ligand is selected
from the group consisting of aminecarboxylates, porphyrins,
cryptates, tetraazacyclododecanes, cyclictetrapyrroles,
aminecarboxylatephosphonates, aminephosphonates, and
phosphates.
11. The method of claim 9, wherein the ligand metabolizable in vivo
is selected from the group consisting of pyrophosphate;
tripolyphosphate, amino acids, pyridoxal, desferrioxamines,
polyglutamic acid, acetoacetic acid, oxalic acid, citric acid,
salicylic acid, acetoacetates, oxalates, salicylates, and citrates;
and the ligand metabolically inert in vivo is selected from the
group consisting of aminecarboxylates,
aminecarboxylatephosphonates, aminephosphonates, porphyrins,
cryptates, tetraazocyclododecanes, cyclic tetrapyrroles, and
phosphates.
12. The method of claim 6, wherein the metabolizable ligand is
tripolyphosphate or pyrophosphate.
13. The method of claim 2, wherein at least one of the ligands is
metabolically inert in vivo.
14. The method of claim 13, wherein the inert ligand is selected
from the group consisting of aminecarboxylates, porphyrins,
cryptates, tetraazacyclododecanes, cyclictetrapyrroles,
aminecarboxylatephosphonates, aminephosphonates, and
phosphates.
15. The method of claim 2, wherein at least one of the ligands is
tripolyphosphate or pyrophosphate and at least one of the ligands
is an aminecarboxylate.
16. The method of claim 15, wherein the aminecarboxylate is
nitrilotriacetate, ethylenediaminetetraacetate,
diethylenetriaminepentaacetate, or
1,4,7,10-tetraazacyclododecane-N,N',N", N'"-tetraacetic acid.
17. The method of claim 2, wherein the complex is adapted to alter
the magnetic resonance properties of sodium ions or water protons
in vivo.
18. The method of claim 2, wherein .sup.r is at least three.
19. The method of claim 2, wherein W is (PPP) and Y is (DTPA),
.sub.m is one, .sub.n is one and .sub.p is zero.
20. The method of claim 2, wherein W is (NTA), Y is (PPP), .sub.m
is one, .sub.n is one and .sub.p is zero.
21. The method of claim 2, wherein W is (NTA) or (EDTA), Y is (PP)
or citrate, .sub.m is one, .sub.n is one and .sub.p is zero.
22. The method of claim 21, wherein M is Dy or Gd.
23. The method of claim 2, wherein the diagnostic method is a
nuclear magnetic resonance imaging procedure, and the paramagnetic
species is employed as a contrast reagent.
24. The method of claim 2, wherein the diagnostic method is a
nuclear magnetic resonance spectroscopic procedure, and the
paramagnetic species in employed as a shift reagent.
25. The method of claim 24, wherein the shift reagent is [Dy(DTPA)
(PPP)].sup.6- or [Dy(ETPA) (PPP)].sup.6-.
26. The method of claim 24, wherein the shift reagent is [Dy(PPP)
(1,4,7,10-tetraazacyclododecane-N, N', N",
N'"-tetraacetate)].sup.6-.
27. The method of claim 2, wherein the complex is adapted to alter
the magnetic resonance properties of sodium ions in vitro.
28. The method of claim 1, wherein the reference ion is a water
proton.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
Paramagnetic species are commonly employed as shift or contrast
reagents in nuclear magnetic resonance (NMR) studies. In clinical
applications, these species are employed to alter magnetic
properties in target tissue to enhance contrast and specificity in
NMR imaging (MRI) and to improve spectrum resolution in NMR
spectroscopy (MRS). Nacked paramagnetic species, however, are
generally of limited clinical relevance in spectroscopic and
imaging procedures, owing to their toxicity.
2. Discussion of Related Art
In order to exploit shift and contrast effects of these
paramagnetic species in clinical diagnostic procedures,
paramagnetic ions such as lanthanides, Fe.sup.3+, Cr.sup.3+, or
Mn.sup.2+ are typically chelated with one or more identical ligands
W to form simple complexes of the type MW.sub.x, wherein x
is.gtoreq.1. Typical ligands W include bidentate and polydentate
ligands such as polyphosphates, especially tripolyphosphate (PPP)
and aminepolycarboxylates such as nitrilotriacetate (NTA),
ethylenediaminetetraacetate (EDTA), and
diethylenetriaminepentaacetate (DTPA). The complexes are structured
according to their intended function: For example, [Dy(PPP).sub.2
].sup.7- is a good shift reagent for sodium spectroscopy as the
favorable geometry of Na.sup.+ relative to the paramagnetic
dysprosium ion [Dy(III)] bound to the highly charged
tripolyphosphate ligand induces large chemical shifts of the sodium
ion; analogously, Gd(III) complexed with PPP to form [Gd(PPP).sub.2
].sup.7- is an effective contrast agent for proton and sodium
imaging.
Unfortunately, many of these simple complexes MW.sub.x known in the
art have limited clinical utility. In the case of
aminepolycarboxylate ligands, the paramagnetic complexes are
typically only effective as contrast reagents in sodium imaging at
relatively high and potentially toxic concentrations, probably
owing to weak binding of Na.sup.+ or unfavorable geometry of the
complex. The tripolyphosphate paramagnetic complexes MW.sub.x are
effective at acceptably low concentrations, but the ligand is
degraded in brain and muscle tissue, most likely by the action of
pyrophosphatase, with resultant deposition of a potentially toxic
paramagnetic ion in the tissue.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 illustrates .sup.31P spectra (121.4 MHz) of aqueous
solutions of: A. 0.2M P.sub.3 O.sub.10.sup.5- ion at pH=7.4,
I=O.sub.3 PO.sub.2 P in PPP.sup.5-, II=PO.sub.2 PO.sub.3 in
PPP.sup.5- ; B. 0.2M [Dy(P.sub.3 O.sub.10).sub.2 ].sup.7-, at
pH=7.4, I=[Dy(PPP).sub.2 ].sup.7-, II=PPP.sup.5- ; C. 0.2M
[Dy(P.sub.3 O.sub.10).sub.2 ].sup.7- and 0.1M P.sub.3
O.sub.10.sup.5- at pH =7.4, I=[Dy(PPP).sub.2 ].sup.7-,
II=PPP.sup.5- ;
FIG. 2 illustrates .sup.31P NMR spectra (121.5 MHz) of aqueous
solutions containing Dy(III), EDTA.sup.4-, and PPP.sup.5- ions;
and
FIG. 3(A) illustrates the .sup.23 Na NMR (79.4 MHz) spectrum of an
erythrocyte suspension made 5.0 mM in [Dy(P.sub.3 O.sub.10).sub.2
].sup.7- : The .sup.23 Na resonance at 2.5 ppm is due to
intracellular Na.sup.+ ion and that at -19 ppm is due to
extracellular Na.sup.+ ion; and
FIG. 3(B) illustrates the .sup.23 Na NMR (79.4 MHz) spectrum of the
erythrocyte suspension of FIG. 3(A) before addition of the complex:
The signal at 0 ppm is the average of extra and intracellular
sodium ion.
SUMMARY OF THE INVENTION
The invention provides anionic mixed complexes of paramagnetic ions
of the formula:
wherein
M is a multivalent paramagnetic ion;
W, Y and Z are each a different polydentate or bidentate ligand
which is a chelator for the ion M;
m+n+p.gtoreq.2 but less than 5 and no more than one of m, n, or p
is zero; and
the complex has an overall negative charge r.
The complexes are useful as contrast and shift reagents for NMR
spectroscopy and imaging procedures, and are particularly useful in
clinical applications in mammals. Owing to the versatility imparted
by the combination of two or three different ligands, the mixed
complexes of the invention are far more effective in a variety of
diagnostic procedures. The ligands W, Y and Z are readily selected
to provide complexes tailored to fit the particular application:
For example, ligands may be selected to increase or decrease tissue
distribution specificity of the complex; to control toxicity of
biodegradation products; to promote accumulation of the
paramagnetic species in target tissue, or to othwerwise vary the
properties of the complexes according to requirements.
DETAILED DESCRIPTION OF THE INVENTION
Paramagnetic species M of the mixed complexes of the invention
broadly include paramagnetic ions having a cationic charge of at
least two, and especially trivalent ions of the lanthanide series,
particularly Dy(III) and Gd(III); other useful paramagnetic ions
include Mn(II), Mn(III), Fe(III), Cu(II) and Cr(III). The
particularly ion M is selected for the intended function of the
complex according to generally accepted standards; for example, M
is selected with reference to the properties of the ligands W, Y
and Z, and with reference to the particular imaging or
spectroscopic technique to be employed. The ions exhibit varying
degrees of specificity and activity as paramagnetic centers of the
complexes of the invention.
The ligands W, Y, and Z are bidentate (having two binding sites to
the paramagnetic species M) or polydentate (having three or more
binding sites to the paramagnetic species M); the net charge r on
the complex comprising the ligands W, Y and, optionally, Z and the
paramagnetic species M is negative, usually greater than (-3). The
complexes are tailored to meet diagnostic requirements: For
example, the ligands can be selected to maximize anisotropy of the
paramagnetism of the ion M to enhance shift and contrast effects of
the paramagnetic species; one or more of the ligands can be varied
in size or lipophilicity or hydrophilicity to diminish or enhance
access to extracellular spaces in vivo; one or more of the ligands
can be selected to be metabolized preferentially by certain cell
types to thereby temporarily accumulate the complex in a target
tissue as a result of altering the net charge on the complex by
elimination of a ligand; one or more of the ligands can be selected
to retard the biodegradability of an otherwise metabolizable
ligand; one or more the ligands can be selected to compensate for a
ligand exhibiting a long term instability in vivo; further, one or
more of the remaining ligands can be selected to a detoxify the
central paramagnetic ion M.
The following guidelines are relevant:
for .sup.23 Na.sup.+ imaging, to produce an observable effect at
very low concentrations of the mixed complexes of the invention,
binding at the sodium site should be strong and the distance
between the Na.sup.+ ion and the paramagnetic ion M should be
minimized to enhance .sup.23 Na.sup.+ relaxation;
for .sup.23 Na.sup.+ spectroscopy, binding should also be strong
but the distance between the paramagnetic M and the Na.sup.+ ion(s)
should be maximized to reduce .sup.23 Na paramagnetic relaxation
and maintain narrow lines for high resolution in the spectrum: The
paramagnetic anisotropy of the mixed complex is generally maximized
to produce a large .sup.23 Na.sup.+ shift at large distances;
for .sup.1 H imaging, any biodegradable ligand should occupy as
large a number of coordination sites as possible consistent with a
strong attachment of a biologically inert (relatively
non-metabolizable) ligand in order to produce an observable effect
at low concentrations of the complex; when the biodegradable ligand
is "lost" to the targeted tissue, water molecules occupy vacated
coordination sites and are subjected to paramagnetic relaxation
enhancement resulting in enhanced contrast in NMR images. In such
an application, the tissue specificity of the complex is
conveniently controlled by selection of the ligand to be one which
is metabolized by target tissue, usually degradation by enzymes
peculiar to the target cells.
In an illustrative example of the invention:
For a complex [MW.sub.m Y.sub.n Z.sub.p ].sup.r- according to the
invention, wherein
M is Dy(III);
W is (PPP).sup.5- (m=1);
Y is (DTPA).sup.5- (n=1); and
p=0; to provide the complex [Dy(PPP) (DTPA)].sup.7- :
(PPP).sup.5- provides a strong .sup.23 Na.sup.+ binding site, and
the illustrated complex [Dy(PPP) (DTPA)].sup.7- is thus
particularly useful as a high resolution agent in sodium
spectroscopy or as an agent for proton imaging of those tissues
capable of causing the decomposition of PPP.sup.5-.
(PPP).sup.5- exhibits a long-term instability in vivo; the
instability is compensated by (DTPA).sup.4-, which is stable in
vivo, and eventually excreted in conjunction with M, thereby
avoiding toxic deposition of M in target tissue;
(PPP).sup.5- is a metabolizable ligand which, in combination with
the inert ligand (DTPA).sup.4-, allows accumulation of the complex
in the target tissue for a sufficiently extended period of time to
permit completion of the NMR diagnostic studies of interest.
Suitable ligands W, Y and Z broadly include those bidentate and
polydentate ligands which function as strong chelating agents for
the selected paramagnetic ion M. Contemplated inactive ligands
(those which tend not to bind sodium or other reference ions when
strongly bound to M) include the class of aminecarboxylates,
exemplified by NTA (nitrilotriacetate), EDTA
(ethylenediaminetetraacetate), and DTPA
(diethylenetriaminepentaacetate); Schiff bases; orthohydroxyphenyl
derivatives; acetylacetone derivatives; template ligands; and
various other polyfunctional amino, hydroxyl and keto compounds;
especially compounds such as porphyrins; 8-hydroxyquinoline;
8-hydroxyquinoline-5-sulfuric acid; aurinetricarboxylic acid;
1,2-bis(salicylideneamino) ethane;
N,N'ethylenedi-(.alpha.-o-hydroxy-phenyl) glycine; hydroxamic acids
and esters thereof, triethylenetetraamine, cryptates, and
tetraazacyclododecanes. Contemplated active ligands (those which
include potential sodium or other reference ion binding sites and
combined to M potentially function as shift and contrast reagents
for sodium and other reference ions) broadly include phosphates,
especially tripolyphosphate and pyrophosphates; citric and aspartic
acid; aminecarboxylatephosphonates; aminephosphonates; small (for
example, C.sub.3-20) peptides with carboxylate side chains; and
oxalates. Ligands potentially metabolizable in vivo broadly include
phosphates such as pyrophosphate (PP) and tripolyphosphate (PPP),
pyridoxal, desferrioxamine, polyglutamic acid, citrates, amino
acids, salicylic acid, acetoacetate, and oxalates. Suitable
esterifying moieties include C.sub.1 -C.sub.6 alkyl groups.
Particular compounds within the scope of the invention which
function to optimize .sup.23 Na and .sup.1 H contrast and shift
effects in MRI and MRS of specific normal and pathologic tissues
include:
[Dy(NTA) (PPP)].sup.5- and [Gd(NTA) (PPP)].sup.5- ;
[Dy(NTA) (PP)].sup.4- and [Gd(NTA) (PP)].sup.4- ;
[Dy(EDTA) (PP)].sup.5- and [Gd(EDTA) (PP)].sup.5- ;
[Dy(EDTA) (Citrate)].sup.4- and [GD(EDTA) (Citrate)].sup.4- ;
and
[Dy(DOTA) (PPP)].sup.6- and [Gd(DOTA) (PPP)].sup.6-.
(where DOTA is
1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid).
Exemplary ligands organized according to function are set forth in
Tables 1 through 3 as follows:
TABLE 1 ______________________________________ POTENTIALLY
BIOLOGICALLY INERT LIGANDS* Ligand Class Examples
______________________________________ Aminepolycarboxylates EDTA,
DTPA, NTA, DOTA Aminephosphonates N(CH.sub.2 PO.sub.3
H.sub.2).sub.3 ; (HO.sub.2 CCH.sub.2).sub.2 NCH.sub.2 PO.sub.3
H.sub.2 Macrocyclic ligands Rifamycin S, porphyrins, tetrapyrroles,
cryptates, 1,4,7,10-tetraazacyclododecane- N, N', N",
N'"-tetraacetic acid Phenolates Ethylenediamine-bis(o-hydroxy-
phenyl) glycine Phosphates Myoinositol hexaphosphate
______________________________________ *These ligands are
potentially useful in metabolizable mixed complexes by retarding
degradation of the metabolizable ligand or by providing a nontoxic
metabolic product comprising a paramagnetic species and the iner
ligand as described supra.
TABLE 2 ______________________________________ LIGANDS WITH
POTENTIAL NA.sup.+ ION BINDING SITES Ligand Class Examples
______________________________________ Aminepolycarboxylates TTHA
(Triethylenetriaminehexa- acetate) Aminephosphonates H.sub.2
O.sub.3 PCH.sub.2 --N(CH.sub.2 CO.sub.2 H).sub.2 Carboxylic acids
Citric, oxalic Phosphates Pyrophosphate, tripolyphosphate,
myoinositol hexaphosphate
______________________________________
TABLE 3 ______________________________________ POTENTIALLY
METABOLIZABLE LIGANDS Ligand Class Examples
______________________________________ Amino acids Aspartic,
aminomalonic Barbiturate Derivatives Aminobarbituric acid - N,N-
diacetic acid ##STR1## Carboxylic acids Citric, salicylic,
acetoacetic, oxalic Hydroxamates Benzhydroxamic acid,
desferrioxamine B Phenolates N-2,3-Dihydroxybenzoylglycine
Phosphates Pyrophosphate, tripolyphosphate, trimetaphosphate,
diphosphoglycerate, phytate.
______________________________________
The mixed complexes of the invention are usefully prepared in
analogous manner to the simple complexes described in the prior
art. Broadly, the mixed complexes are readily prepared by
dissolving an inorganic salt of the paramagnetic ion M
(conveniently the corresponding chloride or oxide) in a first
ligand solution, followed by addition of a second ligand and third
ligand if desired, or by dissolving the salt in a solution of
combined ligands; alternatively, the ligand is dissolved in a
solution of a salt of the ion M, with addition of a second ligand
to the solution, followed by a third ligand if desired. The mixed
complexes are also conveniently prepared by combining solutions of
simple complexes [e.g., (MW).sup.r-,(MY).sup.r- ] or by combining
separate solutions of paramagnetic ion M and individual
ligands.
As will be apparent to those skilled in the art, an extensive
selection of ligands to achieve a variety of results is within the
scope of the invention. Broadly, the complexes are tailored to
optimize chemical shift and/or contrast effects for target tissue
while minimizing or capitalizing upon tissue biodegradation of or
decomposition of a particular ligand depending upon the desired
result, while simultaneously avoiding formation of toxic
by-products. The complexes are tailorable over a broad range of
compositions to optimize .sup.23 NA.sup.+ and/or .sup.1 H contrast
or shift effects in MRI and MRS studies of normal and pathologic
tissues, both in vitro and in vivo, as well as .sup.31 P, .sup.13 C
(termed herein "reference ions") and related spectroscopic and
imaging procedures. Parameters of particular interest for clinical
applications include physiological tolerance (toxicity);
physiological stability (decomposition rate in vivo or in vitro);
nature and strength of the interactions of the complex with water
protons and sodium ions in vivo, and effect on the nuclear magnetic
resonance of water protons and sodium ion in imaging and
spectroscopy on tissues in vivo and in vitro (for .sup.23 Na.sup.+
and .sup.1 H.sup.+ imaging and spectroscopy). The complexes are
generally designed to have a high physiological tolerence; to
effect a larger or smaller region of specific body tissue according
to the influence of the ligands W, Y, Z on the paramagnetism of the
ion M and the translation of this paramagnetism to the water
molecules, sodium ions, or other target; and to accumulate in
different types of tissue according to the ligands selected. The
ligands are usually chosen to selectively accumulate in diseased or
dead tissue cells (infarcts) or in rapidly dividing cells (tumors),
or in normal cells, as desired; the tissue contrast obtained is a
function of tissue ability to accommodate the mixed complexes of
the invention in extracellular or intracellular spaces and to
degrade particular ligands, which in turn is a function of flow,
diffusion, interstitial spaces, lipophilicity or hydrophilicity of
individual ligands and the complex as a whole, enzyme activities,
and other factors. Properly adapted mixed complexes permit
differentiation between normal and diseased tissue, documentation
of methobolic changes induced by radiation damage, hypoxia,
ischemia, and hypoglycemia, evaluation of therapeutic agents on
living tissue, establishment of parameters for normal tissue, an
measurement of a large variety of physiological functions.
The mixed complexes of the invention are employed according to
known prior art procedures; the complexes are typically clinically
administered intravenously or orally, with the amounts administered
being dependent upon the properties of the particular complex, the
target tissue, the specific diagnostic procedure, and other factors
customarily considered in analogous conventional procedures. The
complexes are further useful in non-clinical or laboratory
diagnostic procedures such as those for the differentiation of
tissue in vitro, and other applications. Descriptions of such
procedures are common in the art; exemplary are those set forth in
Lauffer, et al, Magn. Res. Imaging 3:11-16, 1985; Carr, ibid,
17-25, 1985; Runge, et al, ibid, 27-35, 1985; Runge, et al, ibid,
43-55, 1985; and Wesley et al, ibid, 57-64, 1985, all incorporated
herein by reference.
The following Examples are illustrative of the practice of the
invention.
PREPARATION OF SIMPLE AND MIXED COMPLEXES
Example I.
Preparation of complexes by dissolving solid DyCl.sub.3 (hydrated
or unhydrated) in solutions of individual and/or combined
ligands.
A. Dy(P.sub.3 O.sub.10).sup.-2
1. A 0.1M solution was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O in 25 ml of 0.2M P.sub.3
O.sub.10.sup.-5 (see VI.A). While adding the DyCl.sub.3.6H.sub.2 O,
the pH was maintained between 5 and 8. Finally, the total volume
was brought to 50 ml, and the pH was adjusted to 7.
B. [Dy(P.sub.3 O.sub.10).sub.2 ].sup.7-
1. A 0.1M solution was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.H.sub.2 O in 25 ml of 0.4M P.sub.3
O.sub.10.sup.-5 (see VI.E). While adding the DyCl.sub.3.6H.sub.2 O,
the pH was maintained between 6 and 8. Finally, the total volume
was brought to 50 ml, and the pH was adjusted to 7.
C. Dy(EDTA).sup.-1
1. A 0.1M solution was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O in 25 ml of 0.2M EDTA (see VI.B).
While adding the DyCl.sub.3.6H.sub.2 O, the pH was maintained above
6. Finally, the total volume was brought to 50 ml, and the pH was
adjusted to 7.
D. Dy(EDTA).sub.2.sup.-5
1. A 0.1M solution was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O in 25 ml of 0.4M EDTA (see VI.F).
While adding the DyCl.sub.3.6H.sub.2 O, the pH was maintained above
6. Finally, the total volume was brought to 50 ml, and the pH was
adjusted to 7.
E. Dy(EDTA) (P.sub.3 O.sub.10).sup.-6
1. A 0.1M solution was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O in 25 ml of warm 0.2M EDTA (see
VI.B). During the addition, the pH was maintained above 6, followed
by the addition of 1.839 gms (0.005 moles) of Na.sub.5 P.sub.3
O.sub.10. After this the volume was brought to 50 ml while
maintaining the pH at 7.
F. Dy(EDTA) (P.sub.2 O.sub.7).sup.-5
1. A 0.1M solution was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O in 25 ml of warm 0.2M EDTA (see
VI.B). During the addition, the pH was maintained above 6, followed
by the addition of 1.11 gms (0.005 moles) of Na.sub.2 H.sub.2
P.sub.2 O.sub.7. after this, the volume was brought to 50 ml while
maintaining the pH at 7.
G. Dy[N(CH.sub.2 PO.sub.3).sub.3 ](P.sub.3 O.sub.10).sup.-8
1. A 0.1M solutiuon was prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O in 25 ml of 0.2M solution of
nitrilotris (methylene) triphosphonic acid (see VI.D). During the
addition, the pH was maintained above 6, followed by the addition
of 1.839 gms. (0.005 moles) of Na.sub.5 P.sub.3 O.sub.10. After
this, the volume was brought to 50 ml while maintaining the pH at
7.
H. [Dy(EDTA) (P.sub.2 O.sub.7) (P.sub.3 O.sub.10)].sup.-10
1. A 0.1M solution is prepared by dissolving 1.885 gms (0.005
moles) of DyCl.sub.3.6H.sub.2 O and 1.861 gms (0.005 moles) of
disodium ethylenediaminetetraacetic acid dihydrate in 25 ml of 0.2M
P.sub.3 O.sub.10.sup.-5 (see VI A). While adding the salts the pH
is kept between 6 and 8, and then 1.11 gms (0.005 moles) of
Na.sub.2 H.sub.2 P.sub.2 O.sub.7 are added to the resulting
solution. After this the volume is brought to 50 ml while
maintaining the pH at 7.
EXAMPLE II.
Preparation of solutions of complexes by dissolving a ligand in a
solution containing another ligand and/or Dy.sup.3+.
A. Dy(P.sub.3 O.sub.10).sup.-2
1. A 0.1M solution was prepared by dissolving 1.835 gms (0.005
moles) of Na.sub.5 P.sub.3 O.sub.10 in 25 ml of 0.2M solution of
Dy.sup.3+ (see V.A or V.B). The total volume was brought to 50 ml
with distilled H.sub.2 O while adjusting pH to 7.
B. Dy(P.sub.3 O.sub.10).sub.2.sup.-7 or [Dy(PPP).sub.2 ].sup.7-
1. A 0.1M solution was prepared by dissolving 3.679 gms of Na.sub.5
P.sub.3 O.sub.10 (0.01 moles) in 25 ml of 0.2M solution of
Dy.sup.3+ (see V.A or V.B). The total volume was brought to 50 ml
with distilled H.sub.2 O while adjusting the pH to 7.
C. Dy(EDTA).sup.-1
1. A 0.1M solution was prepared by dissolving 1.86 gms (0.005
moles) of Na.sub.2 C.sub.10 H.sub.14 O.sub.8 N.sub.2 2H.sub.2 O
(disodium ethylenediaminetetraacetate dihydrate) in 25 ml of a warm
0.2M solution of Dy.sup.3+ (see V.A. or V.B). The total volume was
brought to 50 ml with distilled H.sub.2 O while adjusting the pH to
7.
D. (Dy(EDTA).sub.2.sup.-5
1. A 0.1M solution was prepared by dissolving 3.722 gms (0.01
moles) of Na.sub.2 C.sub.10 H.sub.14 O.sub.8 N.sub.2 2H.sub.2 O
(disodium ethylenediaminetetraacetate dihydrate) in 25 ml of 0.2M
solution of Dy.sup.3+ (see V.A or V.B). The total volume was
brought to 50 ml with distilled H.sub.2 O while adjusting the pH to
7.
E. Dy(EDTA) (P.sub.3 O.sub.10).sup.-6
1. A 0.1M solution was prepared by dissolving 1.86 gms (0.005
moles) of Na.sub.2 C.sub.10 H.sub.14 O.sub.8 N.sub.2.sup.. 2H.sub.2
O (EDTA) in 25 ml of warm 0.2M Dy.sup.3+ (see V.A or V.B). The pH
of the solution was adjusted to 7, and 1.835 gms (0.005 moles) of
Na.sub.5 P.sub.3 O.sub.10 was added while bringing the total volume
to 50 ml and readjusting pH to 7.
F. Dy(EDTA) (P.sub.2 O.sub.7).sup.-5
1. A 0.1M solution was prepared by dissolving 1.86 gms (0.005
moles) of Na.sub.2 C.sub.10 H.sub.14 O.sub.8 N.sub.2.sup.. 2H.sub.2
O (EDTA) in 25 ml of warm 0.2M Dy.sup.3+ (see V.A. or V.B.). The pH
of the solution was adjusted to 7, and 1.11 gms (0.005 moles) of
Na.sub.2 H.sub.2 P.sub.2 O.sub.7 was added while bringing the total
volume to 50 ml and adjusting pH to 7.
G. Dy[N(CH.sub.2 PO.sub.3).sub.3 ](P.sub.3 O.sub.10).sup.-8
1. A 0.1M solution was prepared by dissolving 2.99 gms of a 50% by
weight solution nitrilotris(methylene)triphosphonic acid in H.sub.2
O in 25 ml of 0.2M Dy.sup.3+ (see V.A or V.B). The pH of the
solution was adjusted to 7, and 1.835 gms (0.005 moles) of Na.sub.5
P.sub.3 O.sub.10 was added while bringing the volume up to 50 ml
and readjusting the pH to 7.
EXAMPLE III.
Preparation of mixed complexes by mixing solutions of individual
pure complexes.
A. Dy(P.sub.3 O.sub.10) (EDTA).sup.-6
1. A 0.1M solution was prepared by mixing 25 ml of 0.2M Dy(P.sub.3
O.sub.10).sub.2.sup.-7 (see IV.B) with 25 ml of 0.2M
Dy(EDTA).sub.2.sup.-5 (see IV.D) while stirring.
EXAMPLE IV.
Preparation of solution of individual complexes by mixing solutions
of Dy.sup.3+ and solutions of individual ligands.
A. Dy(P.sub.3 O.sub.10).sup.-2
1. A 0.1M solution was prepared by mixing equal volumes of 0.2M
Dy.sup.3+ (see V.A or V.B) and 0.2M P.sub.3 O.sub.10.sup.-5
solution (see VI.A). The solution was stirred for one hour.
B. Dy(P.sub.3 O.sub.10).sub.2.sup.-7
1. A 0.1M solution was prepared by mixing equal volumes of 0.4M
P.sub.3 O.sub.10.sup.-5 solution (see VI.E) and 0.2M Dy.sup.3+
solution (see V.A or V.B). The solution was stirred for one
hour.
C. Dy(EDTA).sup.-1
1. A 0.1M solution was prepared by mixing equal volumes of 0.2M
EDTA solution (see VI.B) and 0.2M Dy.sup.3+ solution (see V.A or
V.B). The solution was stirred for one hour.
D. Dy(EDTA).sub.2.sup.-5
1. A 0.1M solution was prepared by mixing equal volumes of 0.2M
Dy.sup.3+ solution (see V.A and V.B) and 0.4M EDTA solution (see
VI.F). The solution was stirred for one hour.
E. Dy[N(CH.sub.2 PO.sub.3).sub.3 ].sup.-3
1. A 0.1M solution was prepared by mixing equal volumes of 0.2M
Dy.sup.3+ solution (see V.A or V.B) and 0.2M
nitrilotris(methylene)triphosphonic acid solution (see VI.D). The
solution was stirred for one hour.
F. Dy(EDTA) (P.sub.2 O.sub.7).sup.-5
1. A 0.066M solution was prepared by mixing equal volumes of 0.2M
Dy.sup.3+ solution (see V.A or V.B) and 0.2M EDTA solution (see
VI.B). This was followed by the addition of an equal amount of 0.2M
P.sub.2 O.sub.7.sup.-5 (see VI.C) solution. The solution was
stirred for one hour.
G. Dy[N(CH.sub.2 PO.sub.3).sub.3 ] (P.sub.3 O.sub.10).sup.-8
1. A 0.066M solution was prepared by mixing equal volumes of 0.2M
Dy.sup.3+ solution (see V.A or V.B) and 0.2M N(CH.sub.2
PO.sub.3).sub.3.sup.-6 solution (see VI.D). This was followed by
the addition of an equal volume of a 0.2M P.sub.3 O.sub.10.sup.-5
solution (see VI.A). The solution was stirred for one hour.
EXAMPLE V.
Preparation of solutions of Dy.sup.3+ in H.sub.2 O
A. From Dy.sub.2 O.sub.3
1. A 0.2M solution was prepared by dissolving 1.492 gms (0.004
moles) of Dy.sub.2 O.sub.3 in 20 ml of 6N HCL and adding H.sub.2 O
to bring the total volume to 20 ml while adjusting the pH to 7.
B. From DyCl.sub.3 (hydrated or unhydrated)
1. A 0.2M solution was prepared by dissolving 3.7695 gms of
DyCl.sub.3.6H.sub.2 O (0.01 mole) in 50 ml of H.sub.2 O while
adjusting the pH to 7.
EXAMPLE VI.
Preparation of solutions of individual ligands.
A. P.sub.3 O.sub.10.sup.-5
1. A 0.2M solution was prepared by dissolving 3.679 gms (0.01
moles) of Na.sub.5 P.sub.3 O.sub.10 in distilled H.sub.2 O. The
total volume was brought to 50 ml while adjusting the pH to 7.
B. EDTA.sup.-4
1. A 0.2M solution prepared by dissolving 3.722 gms (0.01 moles) of
disodium ethylene diaminetetraacetic acid dihydrate (Na.sub.2
C.sub.10 H.sub.14 O.sub.8 N.sub.2 2H.sub.2 O) in warm distilled
H.sub.2 O. The total volume was brought to 50 ml while adjusting
the pH to 7.
C. P.sub.2 O.sub.7.sup.-4
1. A 0.2M solution was prepared by dissolving 2.22 gms (0.01 mole)
of Na.sub.2 H.sub.2 P.sub.2 O.sub.7 (disodium pyrophosphate) in
warm distilled H.sub.2 O. The total volume was brought to 50 ml
while adjusting the pH to 7.
D. N(CH.sub.2 PO.sub.3).sub.3.sup.-6
1. A 0.2M solution was prepared by dissolving 5.98 gms of a 50% by
weight solution of nitrilotris(methylene)triphosphonic acid in
H.sub.2 O, and further dissolving the acid with distilled H.sub.2 O
up to a volume of 50 ml while adjusting the pH to 7.
E. P.sub.3 O.sub.10.sup.-5
1. A 0.4M solution was prepared by dissolving 7.358 gms (0.02
moles) of Na.sub.5 P.sub.3 O.sub.10 in distilled H.sub.2 O and
bringing the volume to 50 ml while adjusting the pH to 7.
F. EDTA.sup.-4
1. A 0.4M solution was prepared by dissolving 7.444 gms (0.02
moles) of disodium ethylenediamine tetraacetate in warm distilled
H.sub.2 O. The total volume was brought to 50 ml while adjusting
the pH to 7.
CHARACTERIZATION OF SIMPLE COMPLEXES OF THE TYPE MW.sub.x
COMPARISON EXAMPLE
EXAMPLE VII.
The Simple Complex, [Dy(PPP).sub.2 ].sup.7- (or [Dy(P.sub.3
O.sub.10).sub.2 ].sup.7-)
A. The .sup.31 P NMR spectra of aqueous solutions of PPP.sup.5- and
[Dy(PPP).sub.2 ].sup.7- ions according to Examples II B and VI A
are illustrated in FIG. 1. As is apparent from the spectra,
coordination of PPP.sup.5- to the paramagnetic Dy(III) ion results
in large downfieldpseudo-contact and contact shifts (+150.1 and
233.1 ppm) of the .sup.31 P resonances which maintain an intensity
ratio of 1:2 when coordinated.
Spectrum 1B is consistent with an equilibrium constant of
-800M.sup.-1 for the following reaction
in which chemical exchange between PPP.sup.5- and [Dy(PPP).sub.2
].sup.7- is slow on the NMR time scale. Spectrum 1C also
demonstrates slow exchange for these entities and provides no
direct evidence for the formation of a [Dy(PPP).sub.3 ].sup.12-
ion. Chemical exchange between PPP.sup.5- and [Dy(PPP).sub.2
].sup.7- with a maintenance of 1:2 ratio of the intensity of the
.sup.31 P resonances of the latter is consistent with the
occurrence of a fluxional process.
B. The addition of [Dy(PPP).sub.2 ].sup.7- from Example VII A
(final concentration, 5 mM) to an erythrocyte suspension (isotonic
choline buffer suspension, 80% hematocrit) results in the immediate
appearance of two Na.sup.+ ion resonances (chemical shift -20. ppm)
in the .sup.23 Na NMR spectra attributable to unshifted
intracellular Na.sup.+ ion and extracellular Na.sup.+ ion which
experiences a pseudo-contact shift via the formation of a weak
complex, {Na[Dy(PPP).sub.2 ]}.sup.6-. These spectra are time
invariant over a 36 hour period (FIG. 3A). In addition to affecting
the chemical shift of the extracellular Na.sup.+ ion, the
[Dy(PPP).sub.2 ].sup.7- agent also changes the relaxation
properties of the Na.sup.+ ion and water. The line width of the
extracellular Na.sup.+ ion changes from -40 Hz (full width at
half-maximum height) in the absence of the reagent to 90 Hz in its
presence. This pronounced effect on the apparent T.sub.2 relaxation
time from 25 milliseconds to 9 milliseconds. The [Dy(PPP).sub.2
].sup.7- shift reagent reduces the T.sub.1 and T.sub.2 values of
extracellular water protons by more than a factor of three. The
precise effect of the agent on the relaxation times is a function
of the ratio of [Na.sup.+ ion]:[agent] and of [H.sub.2
O]:[agent].
CHARACTERIZATION OF MIXED COMPLEXES ACCORDING TO THE INVENTION
Example VIII.
The Mixed Complex, [Dy(EDTA) (PPP)].sup.6- (or [Dy(EDTA) (P.sub.3
O.sub.10)].sup.6-)
A. The .sup.31 P NMR spectra (FIG. 2) of an aqueous solution
containing equal concentrations of Dy.sup.3+ ion and EDTA.sup.4-
ion, and variable amounts of the PPP.sup.5- ion according to
Example III A at pH 7.4 demonstrate that the mixed [Dy(EDTA)
(PPP)].sup.6- complex exists in solution. The relative areas of the
signals assigned to PPP.sup.5- and the [Dy(EDTA) (PPP)].sup.6- ion
are consistent with an equilibrium constant of approximately
20M.sup.-1 for the following reaction at the higher
concentrations
This is contrasted with an approximate equilibrium constant of
>800M.sup.-1 measured by .sup.31 P NMR for the reaction of the
analogous [Dy(PPP).sub.2 ].sup.7- complex
The smaller formation constant for [Dy(EDTA) (PPP)].sup.6- was not
predictable based on gross considerations of charge repulsion
effects. However, more important than overall charge is the effect
on the residual binding capacity of Dy(III) of the partial charge
transferred to Dy(III) in the hexadentate [Dy(EDTA)].sup.- complex
compared to the bidentate [Dy(PPP)].sup.2- complex. Steric and
statistical considerations also affect the relative values of the
formation constants.
B. An isotonic suspension (isotonic choline buffer) of erythrocytes
(80% hematocrit) was made 5 mM with respect to the [Dy(EDTA)
(PPP)].sup.6- ion complex from Example VII A. Two Na.sup.+ ion
resonances appeared in the .sup.23 Na.sup.+ NMR spectrum, with the
extracellular resonance appearing 3.0 ppm upfield from that of the
intracellular Na.sup.+ ion. This is contrasted with a shift of
.sup..about. 20 ppm induced between these two resonances by
[Dy(PPP).sub.2 ].sup.7- at 5 mM (Example VII.B) and with no shift
induced by [Dy(EDTA)].sup.- at concentrations up to 100 mM. The
lack of a [Dy(EDTA)].sup.- ion-induced .sup.23 Na shift is probably
attributable to the weakness of the {Na.sup.+ [Dy(EDTA)].sup.- }
complex and a small anisotropy of the paramagnetic susceptibility
of the [Dy(EDTA)].sup.- complex ion. The smaller .sup.23 Na.sup.+
ion shift of the {Na.sup.+ [ Dy(EDTA) (PPP)].sup.6- }.sup.5-
complex probably resides in both geometric and anisotropic
paramagnetic susceptibility factors. This shift reagent lowers the
relaxation times of extracellular sodium ion by more than a factor
of two, and those of the protons of extracellular water by more
than a factor of five.
EXAMPLE IX.
The Mixed Complex, Dy[N(CH.sub.2 PO.sub.3).sub.3 ] [PPP].sup.8- or
Dy[N(CH.sub.2 PO.sub.3).sub.3 ] [P.sub.3 O.sub.10 ].sup.8-
A. The mixed complex, Dy[N(CH.sub.2 PO.sub.3).sub.3 ] [PPP].sup.8-
in aqueous solution prepared according to Example II G exhibited a
large number of paramagnetically shifted .sup.31 P resonances which
are distinct from the .sup.31 P resonances of the ligands
themselves or the simple complexes Dy[N(CH.sub.2 PO.sub.3).sub.3
].sup.3- and Dy(PPP).sub.2.sup.7-.
B. The addition of this complex (final concentration, 5 mM) to an
erythocyte suspension (isotonic choline buffer, 80% hematocrit)
resulted in the immediate appearance of two Na.sup.+ ion
resonances. The agent reduced the relaxation times of extracellular
Na.sup.+ ion by more than a factor of four and those of the proton
in extracellular water by more than a factor of four.
* * * * *